Experimental Study On Transfecting Red Fluorescence Protein Expressing Vector To The Rat Embryonic Stem Cells By Different Methods

Since Mia Buehr and Ping Li had established rat embryonic stem cells(r ESCs), we have a brand-new animal model for investigating the embryonic stem cells. As rats have many advantages over other murine, they are widely used in Biomedicine. The r ESCs provide us a more superior material to produce genetic rat model via genetically modifying the r ESCs firstly,and play a pivotal role in improving the research method of transgenic technology. We use different methods to transfect p EF1alpha-Ds RedExpress2 Vector into rat embryonic stem cells, and then compare the transfection efficiency. After that, we select the best method to get r ESCs transfected. So we can find an appropriate method to deliver exogenous gene into r ESCs. And the genetically modified r ESCs can be applied in stem cell and xenotransplantation research in our following research.Objective: We used lipofection, electroporation, nucleofection to transfect p EF1 alphaDs Red-Express2 Vector into rat embryonic stem cells. Then we compared the transfection efficiency of different methods and explored whether the pluripotency of r ESCs was changed by the best method with highest efficiency.Methods: The reporter red fluorescent protein(RFP) gene was transfected into r ESCs with different methods. The transfection efficiencies were detected by counting live cells and comparing the cells expressing REP and compared among the different methods. The effect of nucleofection was evaluated by comparing the results of Alkaline Phosphatase(AP) staining, RT-PCR and immunostaining of pluripotency markers of r ESCs before and after nucleofection.Results: The best transfection method was nucleofection. And the highest transfection efficiency was(39.63±1.75)% with the nucleofection program A-13. The results of RT-PCR, immunostaining and AP staining showed that r ESCs maintain the round shape and pluripotency after nucleofection.Conclusion: The nucleofection is an effective method for gene modification of r ESCs compared with lipofection and electroporation.